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In vivo immortalization of murine peritoneal macrophages: a new rapid and efficient method for obtaining macrophage
C Adami1, M J Brunda, A V Palleroni
1Department of Oncology, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Abstract:
Although several murine macrophage (m phi) cell lines from different sites have previously been obtained by in vitro infection with the J2 murine retrovirus, which carries the v-raf and v-myc oncogenes, it was not possible to immortalize thioglycolate-elicited peritoneal macrophages (Pm phi s) by this in vitro procedure. A technique utilizing in vivo injection of the J2 virus has been developed to overcome this problem. The J2 virus immortalized Pm phi s in a very efficient manner in vivo because no exogenous growth factors were required for the in vitro proliferation of these cells and numerous continuous cloned cell lines were readily established. In contrast, Pm phi s obtained from uninfected mice or Pm phi s infected in vitro with the J2 virus did not proliferate. The in vivo immortalized cells had many of the morphological and functional characteristics of m phi s. Analysis of two of the clones, PMJ2-PC and PMJ2-R, demonstrated intracellular expression of the product of the v-raf gene, presence of m phi-associated cell surface antigens, interleukin-6 secretion induced by lipopolysaccharide, and biological response modifier-induced cytotoxic activity against tumor cells. In addition, one of the clones, PMJ2-PC, constitutively expressed major histocompatibility complex (MHC) class II antigens, and in the other clone, PMJ2-R, MHC class II antigens expression was induced by recombinant murine interferon-gamma. This method of utilizing the J2 virus in vivo represents a novel technique for obtaining hematopoietic cell lines from cells that are difficult to immortalize in vitro.
Insights
A novel in vivo retroviral gene transfer method efficiently immortalizes difficult-to-culture murine macrophages (m phi s). This technique overcomes in vitro limitations, establishing continuous cell lines with macrophage characteristics for research applications.
Area of Science:
- * Immunology
- * Virology
- * Cancer Research
Background:
- * Murine macrophage (m phi) cell lines are valuable research tools.
- * Previous attempts to immortalize thioglycolate-elicited peritoneal macrophages (Pm phi s) in vitro using the J2 retrovirus (carrying v-raf and v-myc oncogenes) were unsuccessful.
- * A need exists for efficient methods to generate continuous macrophage cell lines.
Purpose of the Study:
- * To develop a novel technique for immortalizing murine peritoneal macrophages.
- * To establish continuous macrophage cell lines with conserved functional and morphological characteristics.
- * To characterize the immortalized cell lines for research applications.
Main Methods:
- * In vivo injection of J2 retrovirus into mice to immortalize peritoneal macrophages.
- * Establishment and cloning of immortalized macrophage cell lines.
- * Characterization of cell lines for m phi-associated antigens, cytokine secretion, cytotoxic activity, and MHC class II expression.
Main Results:
- * In vivo J2 virus injection efficiently immortalized Pm phi s without exogenous growth factors.
- * Numerous continuous cloned cell lines were established, unlike in vitro infection.
- * Immortalized cells (e.g., PMJ2-PC, PMJ2-R) exhibited m phi morphology and function, including v-raf expression, LPS-induced IL-6 secretion, and tumor cell cytotoxicity.
- * MHC class II expression varied, with constitutive expression in one clone and interferon-gamma inducibility in another.
Conclusions:
- * In vivo retroviral gene transfer is a highly effective method for immortalizing macrophages that are resistant to in vitro immortalization.
- * This novel technique provides a valuable approach for generating continuous hematopoietic cell lines.
- * The established macrophage cell lines serve as robust models for studying macrophage biology and function.